The organ shortage has prompted the use of marginal livers, which are particularly susceptible to ischemia- reperfusion injury (IRI). We discovered that Nrf2 deficiency in the donor liver exacerbates IRI in mouse orthotopic liver transplantation (OLT). However, IRI was prevented if donor livers were conditioned with bone marrow-derived cells overexpressing HO-1. The success of organ rejuvenation was lost after abrogation of SIRT1 activity. Our hypothesis is that SIRT1 signaling in the donor liver: 1/ dictates the rejuvenation outcomes, in which Nrf2 expression represents a denominator of donor liver quality; 2/ serves as a rheostat that controls innate inflammation/regulates hepatocyte regeneration in IR-stressed OLT. We propose three specific aims: Aim 1: Define molecular mechanisms by which SIRT1 signaling controls macrophage inflammation in IR- stressed OLT. Aim 1.1: Test whether activation of macrophage Nrf2 - SIRT1 in the donor liver depresses NF?B signaling/inflammation in IR-stressed OLT. Aim 1.2: Test whether upregulation of SIRT1 in the donor liver depresses the inflammasome platform to block NF?B signaling/inflammation. We will screen for SIRT1- mediated deacetylation of NF?B and expression of SIRT1 regulators (AROS/DBC-1). Then, we will utilize a model of liver IRI in CD11b-DTR mice to dissect SIRT1 - inflammasome cross-regulation in IR-inflammation. Aim 2: Define molecular mechanisms by which SIRT1 signaling regulates hepatocyte regenerative responses in IR-stressed OLT. Aim 2.1: Test whether SIRT1-dependent activation of hepatocyte ?-catenin in the donor liver facilitates cytoprotection in IR-stressed OLT. We will screen for ?-catenin deacetylation under control of SIRT1 regulators (AROS/DBC-1), and then focus on cyclin-D1, and its role in promoting hepatocyte proliferation. Aim 2.2: Test whether SIRT1 activation of parenchyma cell autophagy is required for hepatocyte regeneration in IR-stressed OLT. Two lines of in-vivo (IR-stressed OLT) and in-vitro (H2O2-stressed hepatocyte cultures) studies will assess the significance and mechanisms of enhanced autophagy in SIRT1HI environment (promoting liver rejuvenation) vs. diminished autophagy in SIRT1null environment (sensitizing liver to IRI). Aim 3: Define whether ex-vivo manipulation of Nrf2-SIRT1 axis during hypothermic machine perfusion (HMP) may rescue human discarded livers. Aim 3.1: Test hypothesis that induction of SIRT1 restores function of discarded human livers. Livers deemed unusable will be randomized to: 1/ unmodified HMP; 2/ HMP + SIRT1 activator. The effluent/liver biopsies are analyzed for hepatocellular injury; clearance; biliary function; and hepatic energy status. Aim 3.2: Test hypothesis that hepatic SIRT1 activation rejuvenates human livers. Perfusate/biopsy samples will be screened for the expression of Keap1/Nrf2; SIRT1/inflammasome platform; pro/anti-inflammatory cytokine/chemokine programs; hepatocyte death and ?-catenin/autophagy pathways. Our findings should facilitate innovative donor interventions to improve the quality/size of the current organ supply, an absolute imperative for the full realization of succes for solid organ transplantation.